Chordin-like 1, a Novel Adipokine, Markedly Promotes Adipogenesis and Lipid Accumulation

White adipose tissue serves as a metabolically dynamic organ that can synthesize and secrete biologically active compounds such as adipokines as well as a caloric reservoir for maintaining energy homeostasis. Adipokines are involved in diverse biological and physiological processes and there have been extensive attempts to characterize the effects of over two dozen adipokines. However, many of these adipokines are produced by not only adipose tissue, but also other tissues. Therefore, investigations into the effects of adipokines on physiological functions have been challenged. In this regard, we aimed to identify a new secreted protein that is encoded by genes specifically expressed in white adipose tissue through analysis of multi-tissue transcriptome and protein expression. As a result, we report a novel adipokine that is encoded by the adipose-specific gene, chordin-like 1 (Chrdl1), which is specifically expressed in white adipose tissue in mice; this expression pattern was conserved in the human orthologous CHRDL1 gene. The expression of Chrdl1 was enriched in fat cells and developmentally regulated in vitro and in vivo, and moreover, its retrovirus-mediated overexpression and recombinant protein treatment led to markedly increased adipogenesis. Further pathway enrichment analysis revealed enriched pathways related to lipogenesis and adipogenic signaling. Our findings support a pro-adipogenic role of CHRDL1 as a new adipokine and pave the way toward animal studies and future research on its clinical implications and development of anti-obesity therapy.


Introduction
The global epidemic of obesity is associated with an increased risk of metabolic disorders such as insulin resistance, type II diabetes, hypertension, dyslipidemia, cardiovascular diseases, and cancers [1]. While abnormal accumulation of excessive energy in white adipose tissue (WAT) occurs with obesity, WAT serves, under a normal nutritional state, as a metabolically dynamic endocrine organ that can synthesize and secrete biologically active compounds, including adipokines, as well as a caloric reservoir for maintaining energy homeostasis [2,3]. Expansion of adipose tissue in obesity leads to adipocyte hypertrophy and an increased secretion of pro-inflammatory adipokines, followed by chemokinedriven macrophage accumulation that eventually results in adipose inflammation and insulin resistance [4,5]. Adipose tissue-derived peptide hormones, collectively known as adipokines, are involved in diverse biological and physiological processes and whole-body energy metabolism [6].
There have been extensive attempts to characterize the effects of over two dozen adipokines including the best-known pro-inflammatory leptin and anti-inflammatory adiponectin [7][8][9][10]. However, many of these adipokines are produced by not only adipose tissue, but also other tissues; for example, chemerin has been reported as a hepatokine that is highly produced from the liver as well as an adipokine associated with obesity [11,12]. Therefore, investigations of the effects of adipokines on physiological functions have been challenged. In this regard, further research on secretory proteins that exhibit adipose-specificity and exert its effects on adipogenesis will advance our understanding of the endocrine function of WAT, offering new therapeutic targets for obesity and obesity-related diseases.
In the current study, we report a novel adipokine that is encoded by the adiposespecific gene, chordin-like 1 (Chrdl1). The Chrdl1 gene was specifically expressed in mouse WAT. This WAT-specific expression was conserved in the human orthologous CHRDL1 gene. Further, the expression of Chrdl1 was enriched in fat cells and developmentally regulated in vitro and in vivo. Moreover, retrovirus-mediated overexpression of CHRDL in a preadipocyte cell line led to markedly increased adipogenesis, which was also observed dose-dependently with recombinant CHRDL1 protein. Together with transcriptomebased pathway enrichment results showing enriched pathways related to lipogenesis and adipogenic signaling, our findings support a pro-adipogenic role of CHRDL1 as a new adipokine.

Ethics Statement
All animal care and procedures were approved by the Institutional Animal Care and Use Committees (IACUCs) of The Ohio State University (protocol number: 2007A0183).

Sample Preparation
Total RNA was isolated from tissues and cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as described in our previous reports [13,14]. Stromal vascular (SV) and fat cell (FC) fractions from iWAT harvested from 1-month-old male Friend leukemia virus-B (FVB) mice (n = 3) were separated as described in our previous reports [13]. In brief, the excised tissues were digested in DMEM containing 3.2 mg/mL collagenase II (Sigma-Aldrich) at 37 • C with shaking for 1 h. The digested tissues were then passed through a nylon mesh (100-µm pore size) to eliminate undigested tissue masses and the floating fat cells were separated from the pellet of SV cells by being centrifuged at 500× g for 5 min, then SV and FC fractions were collected for RNA extraction. For RNA isolation for in vivo developmental time-points, 2-, 5-, 10-, 24-, and 49-day-old male FVB mice (n = 3) were subjected to dissection of iWAT. Mouse proteins were extracted from BAT, iWAT, and epididymal white adipose tissue (eWAT) of 3-month-old male FVB mice (n = 3) using lysis buffer as described in our previous report [15]. Human RNAs from the kidney, liver, lung, heart, and muscle tissues were purchased from Agilent Technologies (Santa Clara, CA, USA) and human RNA from WAT was bought from Clontech Laboratories (Mountain View, CA, USA). Human protein extracts from the kidney, liver, lung, heart, muscle, and WAT were obtained from Protein Biotechnologies (Ramona, CA, USA). Samples were stored at −80 • C until use.

Reverse Transcription and Real-Time PCR
These procedures were performed as described in our previous reports [13,16]. In detail, to measure the quantity of RNA, a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used, and approximately 1 µg of RNA was reverse-transcribed in a 20 µL total reaction to cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). The thermal cycle of the reverse transcription was 65 • C for 5 min, 37 • C for 52 min, and 70 • C for 15 min. The quantitative real-time PCR (qPCR) was performed on an ABI 7300 Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) by using AmpliTaq Gold polymerase (Applied Biosystems) with SYBR green detection dye. Forward and reverse primers for Chrdl1 (mF and mR) are listed in Table S1. Reactions were performed in duplicate 25 µL volumes and conditions for the qPCR were 95 • C for 10 min followed by 40 cycles of 94 • C for 15 s, 60 • C for 40 s, 72 • C for 30 s, and 82 • C for 33 s. Relative quantification of gene expression was determined by using the 2 −∆∆CT method [17]. Cyclophilin (Cyc) was used as a housekeeping gene (Table S1).

Western Blot Analysis
Western blot analysis with protein extracts was performed as described in our previous report [16]. Briefly, equal amounts of protein lysates were loaded onto gels before wettransfer to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 30 min and then incubated with an appropriate primary antibody at 4 • C overnight. A CHRDL1 polyclonal antibody was raised against an immunogen (EQVKHSDTYCVFQD) in the rabbit by a custom antibody service (Biomatik; Wilmington, DE, USA) after multiple sequence alignments (Figures S1 and S2). The membranes were probed with antibodies against α-tubulin (12G10, DSHB, lowa City, IA, USA), GAPDH (ab9483, Abcam, Cambridge, MA, USA), HA tag (#2367, Cell Signaling Technology, Danvers, MA, USA), FABP4 (AF1443; R & D systems, Minneapolis, MN, USA), C/EBPα (ARP57937; Aviva Systems Biology, San Diego, CA, USA), and PPARγ (LS-C169542; LSBio, WA, USA). The next day, secondary antibodies were applied to the membrane before developing with ECL plus reagents and X-ray films (both materials from GE Healthcare Biosciences, Pittsburgh, PA, USA).

Cell Culture and Adipogenic Differentiation
The 3T3-L1 preadipocytes were differentiated to adipocytes as previously described (Li et al., 2009). In detail, 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM culture media (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen) and the mixture solution of penicillin and streptomycin (Pen Strep, 100 units/mL; Invitrogen). The preadipocytes were maintained in the culture media and grown to confluence at 37 • C in 5% CO 2 . At 2 days post-confluence (Day 0), differentiation of growth-arrested 3T3-L1 preadipocytes to adipocytes was induced by treating the preadipocytes with differentiation media, which were the culture media supplemented with 0.25 mM isobutylmethylxanthine (IBMX), 1µM dexamethasone, and 1 µg/mL insulin (Sigma-Aldrich Co., St. Louis, MO, USA). Two days after the induction (Day 2), the differentiation media were changed to the insulin media, which contained 1 µg/mL insulin in the culture media and were maintained for the next 2 days. Two days later (Day 4), the insulin media were changed to the DMEM culture media for another 4 days and the media were changed every 2 days. Total RNA was isolated from the 3T3L1 adipocytes at 0-, 2-, 4-, 6-, and 8-day post-differentiation in vitro. In addition, for the recombinant CHRDL1 treatment, mouse CHRDL1 recombinant protein (LS-G25628; LSBio) was purchased and treated for the indicated time with the indicated concentrations (0-400 ng/mL).

Construction of Retroviral Vectors, Viral Transduction, and Differentiation of Stable Cells
The alternatively spliced short-form of the mouse Chrdl1 gene was cloned based on the sequence deposited in the NCBI Gene database (GenBank accession no. NM_031258.3) and tagged with hemagglutinin (HA) in the C-terminal region. Forward and reverse primers (mF and mSR-HA) are listed in Table S1. The cloned isoform with the HA tag was subcloned into the retroviral vector pQCXIP (Clontech) which contains the puromycin resistance gene (Pur r ). The retroviral packaging cell line Ecopack2 TM -293 (Clontech) was transfected with the pQCXIP expression vector with Lipofectamine 2000 (Invitrogen). After 48 h of transfection, the conditioned media containing retroviruses were collected, filtered, snap-frozen, and stored at −80 • C until use. At the time of retroviral transduction, the conditioned media containing virus particles were supplemented to 3T3-L1 preadipocyte culture media. Then, puromycin-resistant stable 3T3-L1 preadipocytes were selected and induced for differentiation. In particular, the stable cells were plated onto 35-mm petri dishes and incubated until they reached 100% confluent. At 2 days post-confluence (Day 0), the cells were treated with a low-concentration adipogenic cocktail [IBMX (62.5 µM), dexamethasone (0.25 µM), and insulin (1 µg/mL), along with the 10% FBS containing DMEM media] to induce slow and minimal adipogenic differentiation. Two days later (Day 2), the cells were treated with the insulin (1 µg/mL) media for 2 more days and then the media were changed at 2 days interval until day 12.

Staining and Imaging
Oil red O (ORO) staining was performed as described in our previous report [18]. In brief, differentiated 3T3-L1 adipocytes were fixed with 10% formalin for 30 m and replenished with 60% isopropanol. Samples were stained with working ORO solution (0.3%) for 10 min at room temperature. After washing with distilled water, the specimens were dried, and photos were taken. Microscopic images of the samples were captured by an imaging system, AxioCam MRc5 (Carl Zeiss, Oberkochen, Germany). The ORO-stained cells were de-stained with isopropanol and the optical density (OD) of the de-staining isopropanol was measured using a spectrophotometer at a 510 nm wavelength.

Transcriptomic Analysis
For recombinant CHRDL1 treatment, total RNAs were isolated from 3T3-L1 cells at day 3 and sent out for sequencing. RNA-seq was performed by the BGISEQ-500 platform, and after read filtering, cleaned reads were mapped to the mouse reference genome (mm10) using HISAT2 [19]. Bioinformatics analysis including Gene Ontology (GO) and KEGG pathway enrichment analysis were performed with processed data.

Data Mining and Processing
Publicly available datasets used in this study were downloaded from the NCBI Gene Expression Omnibus (GEO) database. These datasets include GSE9954 (microarray for various mouse tissues; alternative accession no. GDS3142) [20], SRP020526 (RNA-seq for various mouse tissues) [21], GSE131861 (RNA-seq of mouse BAT, iWAT, and eWAT) [22], and GSE97205 (RNA-seq for human BAT, sWAT, and oWAT). The raw RNA-seq data were processed as described in our previous report [23]. The processed RNA-seq data from various human tissues were derived from the Genotype-Tissue Expression (GTEx) project (GTEx Analysis V7) and further processed as described in our previous report [24].

Statistical Analysis
Gene expression between two means was compared by a two-tailed Student's t-test. To test the normality of the data with multiple means, the Shapiro-Wilk normality test was conducted. For non-normal data, the non-parametric Kruskal-Wallis test was performed followed by the Dunn's post-hoc test. For normal data, one-way ANOVA was conducted followed by the Tukey's post-hoc test. For the tissue distribution of mRNA expression, a mixed ANOVA model followed by a Fisher's protected least significant differences was employed using SAS software version 9.4 (SAS Institute. Inc., Cary, NC, USA).

Expressions of Mouse Chrdl1 Transcripts Were Identified as White Adipose-Specific
To identify a novel gene which is predominantly expressed in adipose tissue, we first sorted 45,101 gene expression profiles as described in our previous report [14] and found microarray probes targeting the last exon of Chrdl1 long-form (1456722_at) and short-form (1434201_at), which were highly ranked as the 277th and 59th adipose-enriched, respectively, out of 45,101 profiles (Table S2). Then, by analyzing RNA-seq data and performing Western blot analysis, the expression of the mouse Chrdl1 gene at both transcript and protein levels was re-examined. Our analysis of RNA-seq data revealed that the mRNA expression levels of both Chrdl1 long-and short-forms (Chrdl1 L and Chrdl1 S, respectively) were significantly higher in white adipose tissue compared to the rest of analyzed tissues ( Figures 1A and S3A). The average TPM values of Chrdl1 long-form (NCBI RefSeq accession no. NM_001358592.1) and short-form (NM_031258.3) were approximately 8-fold and 18fold higher in WAT (subcutaneous white adipose tissue), respectively, than average TPM values of the rest of tissues ( Figure 1A). Next, Chrdl1 expressions in three different adipose depots, i.e., BAT, inguinal WAT (iWAT), and epididymal WAT (eWAT), were analyzed. The expressions of Chrdl1 L and Chrdl1 S were almost imperceptible in BAT, based on RNA-seq read coverages in each exon ( Figures 1B and S3B). In both iWAT and eWAT, the alternatively spliced Chrdl1 S with the premature stop codon (TGA) had an apparently higher expression than Chrdl1 L, based on read coverages on the non-overlapping exons (the Chrdl1 S last exon versus the 10th, 11th, and last exon of Chrdl1 L) ( Figures 1B and S3B). Upon quantification of the normalized read coverages, approximately 3-fold (in iWAT) and 4-fold (in eWAT) higher expressions of Chrdl1 S were shown compared to Chrdl1 L ( Figure 1C). The mRNA levels of markers displayed their depot-enriched trends: brown-fat-selective gene (Ucp1), pan-adipocyte marker (Adipoq), white adipose tissue marker (Lep) [25], supporting proper sample preparation ( Figure 1C). Western blot analysis revealed that the CHRDL1 shortform translation (~37 kDa) occurred prominently in iWAT and eWAT, whereas the CHRDL1 long-form translation was almost absent ( Figure 1D). Taken together, Chrdl1 is a novel WAT-specific gene, and the short transcript (Chrdl1 S) was expressed three to four times more than Chrdl1 L in WAT and was predominantly translated into the CHRDL1 protein.

Expression of the Human CHRDL1 Gene Was Enriched in White Adipose Tissue
For comparative analysis, the expression of the human CHRDL1 gene was further analyzed. By analyzing the GTEx data from human tissue transcriptome studies, a significantly higher expression of CHRDL1 in subcutaneous white adipose tissue was observed than in the rest of tissues (Figure 2A). In three different adipose depots, i.e., BAT, subcutaneous WAT (sWAT), and omental WAT (oWAT), CHRDL1 expression was further

Expression of the Human CHRDL1 Gene Was Enriched in White Adipose Tissue
For comparative analysis, the expression of the human CHRDL1 gene was further analyzed. By analyzing the GTEx data from human tissue transcriptome studies, a significantly higher expression of CHRDL1 in subcutaneous white adipose tissue was observed than in the rest of tissues (Figure 2A). In three different adipose depots, i.e., BAT, subcutaneous WAT (sWAT), and omental WAT (oWAT), CHRDL1 expression was further examined. The expression of the human CHRDL1 transcript (NM_001143981.2) was very low in BAT; however, it was substantially higher in sWAT and oWAT and there were no detectable alternatively spliced transcripts based on RNA-seq read coverages ( Figures 2B and S4). The normalized read coverages were quantified and CHRDL1 expression was approximately 13-fold higher in both sWAT and oWAT than in BAT ( Figure 2C). The brown-fat-selective markers (PRDM16 and PGC1α) were enriched in BAT ( Figure 2C) as well as UCP1 as reported previously [26]. To the contrary, the ITLN1/omentin expression was markedly high in oWAT as expected and ADIPOQ was enriched in sWAT with lesser degrees in oWAT and BAT ( Figure 2C). In addition, the WAT-enriched expression pattern of white adipose tissue marker, LEP, was shown in a previous report [26]. Moreover, Western blot analysis disclosed the CHRDL1 protein expression (~52 kDa) in WAT and its deficiency in other human tissues ( Figure 2D). In summary, despite the absence of an alternatively spliced isoform in the human, the WAT-specific expression is conserved between the mouse Chrdl1 gene and the human CHRDL1 gene at both the transcript and protein levels.

The Chrdl1 Expression Is Enriched in Fat Cells and Regulated during Development In Vitro and In Vivo
To investigate expression levels of Chrdl1 in adipocytes, stromal vascular (SV) and fat cell (FC) fractions were derived from the iWAT of the mouse, and the fractionation was verified by predominant expression of Dlk1 in SV cells, and Fabp4, Scd1, and Pparγ in fat cells (p < 0.05) ( Figure 3A). In SV cells, Chrdl1 mRNA was expressed at low levels. In FCs, the expression level of Chrdl1 mRNA was approximately 2.8-fold higher compared to the level in SV cells (p < 0.05) ( Figure 3A). These expression patterns indicate that the mouse Chrdl1 gene is highly expressed in fat cells (adipocytes). During adipogenic differentiation of the 3T3-L1 preadipocyte cell line in vitro, the Chrdl1 mRNA expression was significantly increased at day 6 post-differentiation and maintained until day 8 ( Figure 3B). Western blot analysis revealed that the mouse CHRDL1 S protein expression was also increased during the differentiation and strong expression signals were detected at day 6 and 8, while the CHRDL1 L protein was unnoticeable ( Figure 3C). To further examine whether the progressive expression pattern of Chrdl1 is also observed in vivo, expression was analyzed with samples from iWAT collected during the development of mice. Expressions of the adipogenic markers, Scd1 and Fas, were significantly increased at day 49 during the pubertal period, and expression of the preadipocyte marker, Dlk1, was significantly decreased at day 5 during the neonatal stage ( Figure 3D). The expression level of Chrdl1 was significantly increased approximately two-fold at day 24 (after weaning) and maintained until day 49 ( Figure 3D). Collectively, in addition to enrichment in fat cells, the expression of Chrdl1 mRNA and CHRDL1 protein increased during adipocyte development in vitro and/or in vivo, suggesting the potential role of the Chrdl1 gene in adipogenesis.
degrees in oWAT and BAT ( Figure 2C). In addition, the WAT-enriched expression pa of white adipose tissue marker, LEP, was shown in a previous report [26]. More Western blot analysis disclosed the CHRDL1 protein expression (~52 kDa) in WAT its deficiency in other human tissues ( Figure 2D). In summary, despite the absence alternatively spliced isoform in the human, the WAT-specific expression is conserve tween the mouse Chrdl1 gene and the human CHRDL1 gene at both the transcrip protein levels.   (Table S3). The interquartile range shows the middle 50% of values. Whiskers indicate variability outside the upper and lower quartiles. Outliers are shown as data points located outside the whiskers. (B) Exon-intron structure of CHRDL1 with the averages of RNA-seq read coverages in BAT, subcutaneous WAT (sWAT), and omental WAT (oWAT) presented in Figure  S4. Transcriptional direction is denoted with an arrow. (C) The mRNA expression levels of CHRDL1, brown-fat-selective genes (PRDM16 and PGC1α), and white adipose tissue markers (ADIPOQ and CFD/ADIPSIN) in sWAT, oWAT, and BAT. Mean TPMs ± SEM are presented. Different letters, a, b, and c, represent statistical difference at a significance level of α = 0.05. (D) Western blot which is representative of two experiments on CHRDL1 protein expression in human tissues. EV, empty-vector control; V+CHRDL1, recombinant vector carrying human CHRDL1 protein; *, non-specific band. The GAPDH protein was used a loading control.

Overexpression of Chrdl1 Increased Adipogenic Differentiation of 3T3-L1 Preadipocytes
To investigate the effect of Chrdl1 on adipocyte differentiation, 3T3-L1 preadipocytes were transduced with recombinant retroviruses producing CHRDL1. For this purpose, retroviruses having a vector fused with HA-tagged Chrdl1 S (S-HA) or an empty vector (EV) were generated using Ecopack2 TM -293 cells followed by Western blotting with an anti-HA tag antibody for confirmation, and the secretion of S-HA was further detected in cell culture media as expected by signal peptide prediction with SignalP-5.0 [27] ( Figures  4A and S1). After the transduction of 3T3-L1 preadipocytes with the retroviruses and selection of puromycin-resistant stable cells, differentiation was induced minimally with the

Overexpression of Chrdl1 Increased Adipogenic Differentiation of 3T3-L1 Preadipocytes
To investigate the effect of Chrdl1 on adipocyte differentiation, 3T3-L1 preadipocytes were transduced with recombinant retroviruses producing CHRDL1. For this purpose, retroviruses having a vector fused with HA-tagged Chrdl1 S (S-HA) or an empty vector (EV) were generated using Ecopack2 TM -293 cells followed by Western blotting with an anti-HA tag antibody for confirmation, and the secretion of S-HA was further detected in cell culture media as expected by signal peptide prediction with SignalP-5.0 [27] ( Figures 4A and S1). After the transduction of 3T3-L1 preadipocytes with the retroviruses and selection of puromycin-resistant stable cells, differentiation was induced minimally with the low-concentration adipogenic cocktail to avoid effects of adipogenic inducers that can override CHRDL1 effect and to obtain maximum contrast in the degrees of differentiation between the two treatments. At day 12 post-differentiation, S-HA overexpressing cells displayed markedly greater lipid accumulation (approximately 6-fold higher) and a significantly increased number of adipocytes in Oil Red O staining and phase contrast microscopy, respectively, compared to EV transfected cells ( Figure 4B). The adipogenic differentiation was confirmed by PPARγ and FABP4 protein expression at Days 8 and 12 post-differentiation ( Figure 4C). The S-HA overexpressing cells exhibited higher PPARγ and FABP4 protein expression levels than EV transfected cells, indicating greater adipogenic differentiation. Of note, the S-HA protein expression level was relatively consistent during differentiation, and it was due possibly to the effect of a strong CMV promoter in the retroviral vector ( Figure 4C). Overall, the mouse CHRDL1 short-form overexpression resulted in an increased adipocyte differentiation and lipid accumulation, implying the role of CHRDL1 as a candidate pro-adipogenic adipokine.

Recombinant CHRDL1 Protein Increased Both Induced and Spontaneous Adipogenic Differentiation Dose-Dependently
For the purpose of determining a permissive stage for adipogenic differentiation by CHRDL1 and its dose-dependent effects, recombinant CHRDL1 protein was administered to the 3T3-L1 cell media. By comparing different periods for CHRDL1 recombinant protein treatment (100 ng/mL) which were either throughout, prior to, or after the time of differentiation induction (D0) (Figure 5A), the permissive stage for adipogenic differentiation by CHRDL1 was ascertained. Among the treatments 1 through 3, treatment 3 with both differentiation induction and CHRDL1 recombinant protein showed much more lipid accumulation and expression of adipocyte differentiation marker protein (FABP4) at day 8 (D8) ( Figure 5B). Then, the period of treatment 3 was divided into periods for the treatment 4 and 5, and, between treatment 4 and 5, substantially more lipid accumulation and expression of FABP4 were observed with treatment 5, suggesting a stimulatory effect of CHRDL1 recombinant protein on adipocyte differentiation after the induction of differentiation at D0 ( Figure 5B). To narrow it down, the period of treatment 5 was divided into periods for treatments 6 through 8, and, among treatments 6-8, treatment 6 and 7 showed considerably higher levels of lipid accumulation and FABP4 expression, implying the period of D0 through D4 is the permissive stage of 3T3-L1 differentiation by recombinant CHRDL1 protein ( Figure 5B). Therefore, at 4 days of differentiation, the treatment during the permissive stage (D0-D4) with four different concentrations of recombinant CHRDL1 protein were evaluated, and it was shown that the lipid accumulation increased very strongly depending on the dose ( Figure 5C). Further, the expression adipocyte differentiation markers (PPARγ, C/EBPα, and FABP4) increased markedly and dose-dependently ( Figure 5D). Together, it suggested that CHRDL1 mediated such a strong and fast promotion of adipogenesis, which has never been reported to the best of our knowledge. On a next evaluation, spontaneous differentiation with higher concentrations also led to an increased adipogenic differentiation in a dose-dependent manner, consolidating the potential pro-adipogenic role of CHRDL1 ( Figure 5E,F). In conclusion, the effects of CHRDL1 on adipocyte differentiation were assessed temporally and dose-dependently, providing evidence of a strong stimulatory effect of CHRDL1 on adipogenesis.
post-differentiation ( Figure 4C). The S-HA overexpressing cells exhibited higher PPARγ and FABP4 protein expression levels than EV transfected cells, indicating greater adipogenic differentiation. Of note, the S-HA protein expression level was relatively consistent during differentiation, and it was due possibly to the effect of a strong CMV promoter in the retroviral vector ( Figure 4C). Overall, the mouse CHRDL1 short-form overexpression resulted in an increased adipocyte differentiation and lipid accumulation, implying the role of CHRDL1 as a candidate pro-adipogenic adipokine.

Transcriptomic Analysis Revealed That CHRDL1 Up-Regulates Signaling Pathways Related to Adipogenesis
To further investigate whether CHRDL1 is related to cell signaling pathways regulating adipogenesis, we performed RNA-seq using samples prepared with or without the recombinant CHRDL1 treatment (100 ng/mL) and on day 3 when the 3T3-L1 cells were differentiating after mitotic clonal expansion (two rounds of mitosis) for 2 days post differentiation induction [28] (Figure S5). Among 16,608 analyzed genes, there were 33 up-regulated differentially expressed genes (DEGs) and 3 down-regulated DEGs, with a threshold of an absolute value of fold change > 2 and adjusted p-value < 0.05 ( Figure 6A, Table S4). The KEGG pathway analysis showed that a signaling pathway with the highest rich factor (enrichment factor) was 'Fatty acid biosynthesis' which consisted of genes encoding acyl-CoA synthetases (Acsl1 and Acsbg1; both were up-regulated.) ( Figure 6B). Further, a signaling pathway with the lowest q-value (3.48 × 10 −11 ) was the 'PPAR signaling pathway', which is composed of all up-regulated genes (Adipoq, Fabp4, Cd36, Cpt1a, Acsl1, Hmgcs2, Fabp5, Angptl4, Acsbg1, and Plin1) that are involved in adipogenesis ( Figure 6B). Generally, the lesser the enrichment factor, the higher the q-value (lesser significance). In addition, gene ontology (GO) analysis showed that many up-regulated DEGs are involved in cellular developmental processes ( Figure 6C). In summary, the most significantly enriched pathways under CHRDL1 treatment were related to lipogenesis and adipogenic signaling.

Transcriptomic Analysis Revealed That CHRDL1 Up-Regulates Signaling Pathways Related to Adipogenesis
To further investigate whether CHRDL1 is related to cell signaling pathways regulating adipogenesis, we performed RNA-seq using samples prepared with or without the recombinant CHRDL1 treatment (100 ng/mL) and on day 3 when the 3T3-L1 cells were differentiating after mitotic clonal expansion (two rounds of mitosis) for 2 days post differentiation induction [28] (Figure S5). Among 16,608 analyzed genes, there were 33 up- pathway', which is composed of all up-regulated genes (Adipoq, Fabp4, Cd36, Cpt1a, Acsl1, Hmgcs2, Fabp5, Angptl4, Acsbg1, and Plin1) that are involved in adipogenesis ( Figure 6B). Generally, the lesser the enrichment factor, the higher the q-value (lesser significance). In addition, gene ontology (GO) analysis showed that many up-regulated DEGs are involved in cellular developmental processes ( Figure 6C). In summary, the most significantly enriched pathways under CHRDL1 treatment were related to lipogenesis and adipogenic signaling.

Discussion
Here, we report a newly identified adipokine CHRDL1 that is encoded by the white adipose-enriched Chrdl1 gene. Through extensive comparison of various mouse and human tissues using transcriptome analysis and Western blot analysis, the WAT-enriched expression pattern of Chrdl1, the orthologous human CHRDL gene, and their encoded protein CHRDL1 were revealed, suggesting their potential roles in adipocyte biology. Both mouse and human CHRDL1 were detected as a single dominant isoform in WAT. Noticeably, unlike Adipoq which is expressed in both white and brown adipocytes [29], expressions of the Chrdl1 gene and its encoded protein CHRDL1 was almost undetectable in BAT. Further, unlike ITLN1 (omentin-1) which is specific to omental fat, but not subcutaneous fat, in the human [30], CHRDL1 expression levels were significantly high in both oWAT and sWAT while undetectable in BAT. Identifying the adipose tissue-enriched genes that are expressed predominantly in adipose tissue and further determining secreted proteinencoding genes among those genes have been conducted during candidate novel adipokine research [7][8][9]30]. In addition, regarding adipocyte differentiation, a slight enhancement has been reported with adipokine vaspin treatment [31]. However, as reported in this study, detection of the strong and fast promotion of adipogenesis will be important for targeting critical adipokines for downstream analysis.
As 3T3-L1 preadipocyte cell lines are considered an appropriate in vitro model for investigating adipocyte secretome [32], the secretion of CHRDL1 to the 3T3-L1 cell culture media was confirmed in this study. The previous estimate of total number of proteins in secretome reached approximately 2200 which is about 7% of a total 30,000 human proteins and many secretory proteins are expressed from multiple tissues and organs [33,34]. Considering the intricacies of multi-organ systems, developing tools for identifying tissuespecific secretory proteins is of particular interest and ongoing [35]. Because Chrdl1 is specifically expressed in WAT, investigations on the effects of secreted CHRDL1 will advance our understanding on adipokines predominantly derived from WAT. Further, the developmental expression pattern described above showed that the CHRDL1 expression was gradually increased during the differentiating stage of the 3T3-L1 preadipocytes and maintained at a fully differentiated stage (mature adipocytes), implying its role during adipocyte development. Retrovirus-mediated overexpression of CHRDL1 in the 3T3-L1 preadipocyte cell line and recombinant CHRDL protein treatments provided evidence for the previously unrecognized effect of CHRDL on adipogenesis. The CHRDL1 produced in adipocytes may affect adipocytes themselves and/or neighboring cells in autocrine and/or paracrine manners, or CHRDL1 from mature adipocytes may trigger adipogenic differentiation of nearby preadipocytes which might be potential action mechanisms in adipose tissue in vivo.
The pathogenic effects related to obesity are greatly attributed to dysregulated secretion of adipokines, which leads to decreased insulin sensitivity (insulin resistance) in peripheral tissues including skeletal muscle and the liver [4]. Therefore, adipokines are promising candidates for the future treatment of obesity and obesity-related diseases. As indicated in the aforementioned pathway analysis, acyl CoA synthetases were up-regulated with CHRDL1 treatment and could convert free fatty acids to fatty acyl-CoA esters, and thereby play a key role in lipid biosynthesis [36]. Interestingly, reduced concentration of plasma lipids was recently reported to be strongly correlated with increased CHRDL1 expression [37]. Also, supplementary data from a previous study on sex and aging plasma proteomes showed that mouse plasma CHRDL1 did not change with sex whereas it was significantly up-regulated with age (q = 8.57 × 10 −4 ) [38] This increased circulating level of CHRDL1 during aging was conserved in humans (q = 8.36 × 10 −38 ) and consistent with previous results [39]. In this regard, interactions between CHRDL1 levels, age, and plasma lipids that might affect cardiometabolic health should be further investigated. In addition, as experimental perspectives, for the induction of differentiation, a reduced concentration of IBMX and dexamethasone was used to show a more drastic effect of CHRDL on the differentiation, and it led to very low lipid accumulation in control cells and much higher lipid accumulation in CHRDL1 overexpressing cells. In addition, the HA-tag was used to distinguish endogenous CHRDL1 expression and overexpressed CHRDL expression. Further, for future research, expressions of binding partners in white adipose tissue (autocrine/paracrine signaling) or other tissues (endocrine signaling) need to be identified and analyzed. Overall, our findings support the pro-adipogenic role of CHRDL1 as a new adipokine and pave the way toward animal studies and future research on its clinical implications and development of anti-obesity therapy.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells12040624/s1, Figure S1: Amino acid sequence analysis of CHRDL1; Figure S2: Multiple alignment of chordin-like 1 nucleotide sequences; Figure S3: Expression levels of mouse Chrdl1 mRNA; Figure S4: Expression levels of human CHRDL1 mRNA; Figure S5: Measurement of cell numbers; Table S1: Primer sequences for real-time PCR and cloning; Table S2: Microarray-based mouse transcriptome dataset; Table S3: Sample sizes of the GTEx data for human tissues. Table S4: RNA-seq analysis of 3T3-L1 cells with or without CHRDL1 treatment.

Data Availability Statement:
The data generated and processed during this study can be found within the manuscript and Supplementary Materials. Publicly available datasets processed in this study include GSE9954 (microarray for mouse tissues), GSE132040 (RNA-seq for 3-month-old mouse tissues), GSE131861 (RNA-seq for mouse BAT, iWAT, and eWAT), GSE97205 (RNA-seq for human BAT, sWAT, and oWAT), and GTEx Analysis V7 (processed RNA-seq data for human tissues).